1. Field of the Invention
The present invention relates to an improved arthropod diet packaging, presentation, and delivery system for the feeding or oviposition of arthropods. In particular, the invention is directed to an improved diet delivery system having a diet-filled reservoir covered with a stretchable membrane, wherein the reservoir further has diet-filled outwardly projecting protrusions formed in the stretchable membrane, which protrusions are in fluid communication with the reservoir. The invention is also directed to methods of making the diet delivery system of the invention. The arthropod diet delivery system is suitable for mass production of arthropods at a reasonable cost for uses including as biological control agents.
2. Description of the Art
Biological control is recognized as one of the best alternatives to the use of chemical insecticides for controlling insect pests. Use of beneficial arthropod predators and parasites for biological control on a large scale as an alternative to pesticides depends on the ability to mass produce large quantities of viable and biologically fit arthropods at a reasonable cost. Rearing of beneficial arthropods on their natural hosts/prey or on unnatural factitious hosts is too expensive to allow large scale use of beneficial arthropods in commercial agriculture. Accordingly, artificial diets or growth media and effective diet packaging/presentation/ delivery systems are required for mass production at reasonable cost.
The Review Article by A. C. Cohen, D. A. Nordlund and R. A. Smith, xe2x80x9cMass Rearing of Entomophagous Insects and Predaceous Mites: Are the Bottlenecks Biological, Engineering, Economic, or Cultural?, xe2x80x9d Biocontrol News and Information, 20 (3): 85N-90N (1999) provides a detailed discussion of barriers to overcome in successful mass rearing of natural enemies. These include: development of nutritionally adequate artificial diets, suitable packaging for the diets, nondestructive handling of insects and mites, adequate moisture, prevention of microbial contamination of diet or insects living in close association prevention of loss of genetic fitness, adequate ventilation, appropriate thermal conditions, appropriate lighting, accommodation for moulting, oviposition sites, and accommodation for newly-closed first instars that are extremely vulnerable to desiccation and starvation.
High quality semi-solid artificial diets for mass rearing of arthropods at a reasonable cost have been described. U.S. Pat. Nos. 5,834,177 and 5,945,271 to Cohen describe semi-solid artificial diets for rearing entomophages (predatory arthropods and parasitic insects) which comprise a mixture of (a) cooked whole egg, (b) a protein-lipid paste (e.g., a mixture of ground beef and beef liver), and (c) a liquid, wherein the cooked whole egg forms a sticky, stringy substrate that keeps the mixture in stable form. The diet provides nutrients effective for supporting the growth of larvae of entomophages from the time of hatching until pupation and also provides nutrients effective for rearing entomophages which have predaceous adult stages. This diet parallels the texture and consistency of the insides of the natural prey, and is particularly suitable for rearing insect predators that pre-digest their prey and must recapture their digestive enzymes and ingest the digested, liquified medium to complete their digestion (extra-oral digestion). The diet may be used as a fresh semi-solid diet mixture or as a freeze-dried and reconstituted semi-solid diet. The diet may also be used as a supplement in the artificial diet for phytophagous (plant-eating) insects that supplement their plant-eating habits with insect consumption. Exemplary entomophages reared on the growth media include Chrysoperla carnea Stephens/Chrysopidac (lacewings); Geocoris punctipes (Say)/Lygaeidae (big eyed bug); Serangium parcesetosum; Orius insidiosus; and Perillus bioculatus and Podisus maculiventris (which are both predatory stink bugs in the family Pentatomidae).
A. C. Cohen and L. K. Smith (Biological Control 13:49-54 (1998)) report production of 15 continuous generations of green lacewings, Chrysoperla rufilabris Burmeister (Neuroptera: Chrysopidae), using the semi-solid diet of U.S. Pat. No. 5,834,177.
U.S. Pat. No. 6,235,528 B1 to Cohen describes semi-solid artificial diets or arthropods, including zoophagous arthropods and phytophagous arthropods including facultatively zoophagous phytophages. In one aspect, the growth medium is composed of a mixture of cooked egg, liquid, and carbohydrate source. In a second aspect, the growth medium is composed of a plant-based phytophage diet which includes cooked egg yolk or cooked whole egg. In a third aspect, the growth medium is composed of a mixture of cooked egg, liquid, and carbohydrate source in admixture with a plant-based phytophage diet which includes cooked egg yolk or cooked whole egg. The diets may be used as fresh or freeze-dried and reconstituted semi-solid diets. Exemplary arthropods fed on a selected diet included the entomophage Chrysoperla rufilabris (green lacewling) and Lygus hesperus Knight and Lygus lineolaris (facultatively zoophagous phytophages)
Semi-solid meat-based artificial diets have been described for rearing Geocoris punctipes (Say) in publications by A. C. Cohen, Journal of Economic Entomology, 78:1173-1175 (1985); A. C. Cohen and N. M. Urias, The Southwestern Entomotologist, 11:171-176 (1986); and A. C. Cohen and R. T. Staten in Applications of Genetics to Arthropods of Biological Control Significance, Eds. S. K. Narang et al., CRC Press, Inc., Chapter 7, pp. 121-132 (1994)). De Clercq et al. (Entomophaga 37:149-157 (1992) and Biological Control 12:137-142 (1998)) describe an artificial insect diet for rearing the predatory stinkbugs Podisus maculiventris and Podisus sagitta using the meat-based diet of Cohen (1985) with added fresh (raw, liquid) egg yolk. Saavedra et al. (Med Fac Landbouww Univ Gent61(3a):767-772 (1996) describe an artificial insect diet for Podisus nigrispinus based on the bovine meat diet developed by Cohen (1985, supra) having added bee""s honey, brewer""s yeast, fresh egg yolk, and Wesson""s salt.
The problem of packaging an artificial arthropod diet is complicated by the need, in most cases, to maintain both moisture and a barrier to microbial attack while still keeping the diet accessible and phagostimulatory to the insects (Cohen et al., 1999, supra). Standard techniques for packaging diets for presentation and delivery to arthropods include packaging the diet in a membrane such as Parafilm(copyright) (a flexible, moldable self-sealing, odorless, moisture resistant, thermoplastic, semi-transparent, and practically colorless membrane). The packaged medium can be sterilized and will remain sterile for subsequent use for rearing the target arthropod.
The packaged diet can be presented to the arthropods in a shape and wall thickness that simulates natural prey. Cohen, 1985, supra, and Cohen and Urias, 1986, supra, report good production of larvae with a diet delivery system using a single layer of Parafilm(copyright) (stretched to 3-fold its normal width) wrapped around the meat-based semi-solid diet in cylindrical form and pressure-scaled along the longitudinal seam. Cohen and Staten, 1994, supra, report using flattened packets made of stretched Parafilm to contain the diet.
Cohen and Smith, 1998, supra, report feeding packets formed by sandwiching the semi-solid diet in two Parafilm(copyright) layers that were stretched to about three times their original length and width (i.e., as 45-micron-thick membranes). The membranes were scaled around the diet to form a flat sachet. U.S. Pat. No. 5,834,177 reports using Parafilm(copyright) stretched to about 15-20 microns thick.
Packaging of small volumes of liquid artificial diets in a thin membrane or coating using encapsulation or coating techniques, for use in rearing or oviposition of predators of harmful insects has been reported. In these methods, a capsule, a hemispherical well in a stretched membrane or other container for holding the liquid diet is formed and is subsequently filled with an aliquot of a liquid nutritive growth medium or liquid inducement medium. As necessary, air bubbles in the liquid are then removed, for example, with a syringe. The container is then sealed.
K. S. Hagen and R. L. Tassan (Journal of Economic Entomology 58:999-1000, 1965) describe a technique for producing paraffin-coated diet capsules to supply artificial liquid diet to Chrysopa larvae. A. C. Cohen (Journal of Economic Entomology, 76(4):957-959, 1983) describes improved methods for encapsulating small units of liquid suspension artificial diets in mixtures of waxes and plastic polymers, for rearing predators for use in biological control of insect pests. A. N. Dahlan and G. Gordh (Entomophaga 42(4):525-536, 1997) describe artificial eggs formed of an upper cover of clear polypropylene, 32-36 microns thick and a lower cover of clear polyethylene, 8-13 microns thick and covered with gelatine solution. Each egg was filled with 5-6 microliters of diet solution for development of Trichogramma australicun Girault.
Z.-H. Xie et al. (Biological Control 8:107-110, 1997) describe in vitro culture of Trichogramma spp. on artificial liquid diets contained in stretched plastic artificial eggs prepared with polyethylene film and coated with Elmer""s School Glue (Borden, Inc., Columbus, Ohio) (20 microliters diet/stretched plastic artificial egg). D. A. Nordlund et al. (Biological Control 9:201-207, 1997) describe wax artificial eggs (WAEs) which consisted of liquid droplets of diet coated with a thin layer of paraffin-Vaseline(copyright) mixture (75%:25%) which were used to induce Trichogramma minutum Riley females to oviposit. S. M. Greenberg et al. (Biological Control 11:43-48, 1998) describe a study of the oviposition response of adult Trichogramma minutum Riley and Trichogramma pretiosum Riley females to wax artificial egg (WAEs) and stretched plastic artificial eggs (SPAEs) with 0.01% sodium bisulfite solution as an oviposition stimulant. The WAEs had a mean diameter of 3.2xc2x10.03 mm. The SPAEs were prepared with a polypropylene film (22.9 microns thick) by impressing, by hand, the surface of the film with a 2-mm diameter and 1.5-mm high peg. The film was held, by vacuum, on a perforated metal female mold with 3.175-mm holes. After the SPEAs were formed (70 per 16 cm2 area), the vacuum was released and the polypropylene film was removed and placed in the oviposition arena. The SPAEs were held convex side to the Tichogramma. The oviposition arena was then filled with oviposition stimulant. Any air bubbles that formed in the SPEAs were removed with a syringe.
J. E. Carpenter and P. D. Greany (Biological Control 11:203-208 (1998)) describe a liquid diet encapsulation method wherein Parafilm(copyright) was stretched bidirectionally to a final thickness of ca. 10-15 microns and applied to a template (made by drilling 24 1-cm diameter holes on 2-cm centers in a 4xc3x976 pattern into a 1-cm aluminum plate). This plate was attached to a plenum, so that a vacuum could be applied after the Parafilm(copyright) was stretched over its surface. Hemisperical depressions (wells) were formed in the Parafilm(copyright) using a slight negative pressure (xe2x88x925.0 cm mercury) along with mechanical distortion achieved by applying a probe slightly smaller in diameter than the hole in the template. The probe was pressed into the Parafilm(copyright), stretching it to the near-breaking point, forming a well of about 0.5 ml in volume. The liquid medium was homogenized and strained through a wire mesh of ca. 2xc3x972 mm, then pipetted into the wells, which were then sealed by applying a plastic film such as Handiwrap(copyright) to the surface of the template. A piece of aluminum foil was applied over the plastic wrap and a heated brass roller (ca. 170xc2x0 C.) was used to heat seal the Parafilm(copyright) to the plastic wrap. The final encapsulated diet sheets resembled xe2x80x9cbubble pakxe2x80x9d used in packaging, that is, the individual wells of diet were surrounded by sealed film. U.S. Pat. No. 5,799,607 to Greany and Carpenter also describe this encapsulation procedure of a liquid medium.
U.S. Pat. No. 4,418,647 to Hoffman describes an artificial membrane having a curvilinear surface region, the outer surface of which is adapted to induce oviposition by adult Trichogramma wasps, and the inner concave surface of which defines a semi-enclosed cavity adapted for recovery of the oviposited eggs.
In the foregoing methods of packaging small volumes of artificial liquid diets in coated or encapsulated form for rearing or oviposition of insects, the container for the aliquot of liquid diet, e.g., well or capsule, was pre-formed and then filled with the liquid diet or inducement liquid in a separate subsequent step. Because liquids readily flow and seek their own level, the packaging of small volumes of liquid diets is relatively straight forward. The liquid diet will fill a container, filling in each nook and cranny. Air bubbles therein can be readily removed.
This is not true of semi-solid arthropod diets. Because of the thick, viscous properties of semi-solid arthropod diets, filling small volumes, e.g., less than about 0.5 cm diameter, has been heretofore impracticable. Presently, semi-solid diets are presented in larger volumes, e.g., diet packets of about 1xc3x971xc3x970.5 cm; 10.16xc3x9715.24xc3x970.5 cm; 10.16xc3x9750.8xc3x970.5 cm, which are covered or enclosed with a membrane. Covering the semi-solid diet with a thin membrane requires tedious stretching of the membrane by hand prior to enclosing the diet in the membrane. This time-consuming and labor intensive procedure of hand stretching of a membrane is not susceptible to automation, and because of the labor intensity, it adds to the cost of packaging arthropod diets.
As discussed above, artificial semi-solid arthropod diets have proven to be highly valuable for producing large numbers of viable and biologically fit arthropods for uses including as biological control agents. What is needed is an efficient system for packaging small volumes of a semi-solid arthropod diet in a stretched, thinned membrane.
The present invention is directed to an improved system for the packaging, presentation, and delivery of a semi-solid arthropod diet, for the feeding or oviposition of arthropods, hereinafter denoted as arthropod diet delivery system. The arthropod diet delivery system of the invention comprises a diet-filled reservoir covered with a stretchable membrane, wherein the reservoir membrane has a plurality of arthropod feeding protrusions thereon which comprise stretched, thinned outwardly projecting areas in the stretchable membrane. The protrusions are filled with the semi-solid diet, and the interior of a protrusion is in fluid communication with the diet-filled reservoir.
The arthropod diet delivery system may optionally further include stretched, thinned inwardly protecting areas in the stretchable reservoir membrane, denoted as intrusions. The intrusions are particularly advantageous as oviposition sites for certain arthropods.
The invention is also directed to methods of making the arthropod diet delivery system wherein the protrusions are substantially simultaneously created and filled with diet. In these methods, the process of stretching the membrane to form protrusions and filling the protrusions comprise one step. These methods are amenable to scale-up and to automated packaging techniques. They are especially amenable to production of small, filled protrusions, which are difficult to attain by systems that are designed to fill pre-formed protrusions.
In brief, methods of making the arthropod diet delivery system of the invention comprise acting on a stretchable membrane which covers or encloses a reservoir of semi-solid arthropod diet with a protrusion-creating implement in a manner to create a plurality of stretched, thinned outwardly projecting areas in the reservoir membrane, denoted as protrusions, and to substantially simultaneously fill the protrusions with diet. Optionally, the implement may also create intrusions in the reservoir membrane. The result is a diet delivery packet having the desirable properties of selective thinning of the membrane material at the sites of potential feeding or oviposition, while protecting the diet from degradative exposure of the diet to the environment.
The semi-solid arthropod diet delivery system of the invention fulfills an important need for an effective diet packaging, presentation, and delivery system for efficient mass rearing of arthropods. Millions of arthropods are required for augmentative biological control and other biologically based technologies. Obtaining very large quantities of arthropods demands both (1) the use of inexpensive, highly nutritious diets which yield large numbers of viable and biologically fit arthropods at reasonable cost, such as the artificial semi-solid diets described above, and (2) an effective diet packaging, presentation, and delivery system for these artificial diets, which system is amenable to scale-up and to automated packaging techniques. The arthropod diet delivery system of the invention fulfills this need of providing an economical means for rearing arthropods. The invention also fulfills the need for large scale production of arthropods necessary for technologies such as augmentative biological control, sterile insect release, and production of pathogens and parasitic insects using mass-produced arthropods as food.
The present invention provides an efficient system for presenting arthropods with small volumes of semi-solid diet in a stretched, thinned membrane, e.g., protrusions, while avoiding the time-consuming and labor intensive procedure of hand stretching of a membrane. Additionally, because the diet within a protrusion is in fluid communication with the diet-filled reservoir, the invention diet delivery system provides arthropods with access to an abundance of diet medium.
Accordingly, it is an object of the invention to provide an improved system for the packaging, presentation, and delivery of semi-solid arthropod diet for the feeding or oviposition of arthropods and methods of producing the same.
Another object of the invention is the provision of an effective and economical diet delivery system for the large-scale production of arthropods.
A further object of the invention is the provision of a system for the packaging and delivery of semi-solid diets which system is amenable to scale-up and suitable for automated packaging techniques.
A still further object of the invention is to provide protection of arthropod diet from desiccation and microbial contamination.
Other objects and advantages of the invention will become readily apparent from the ensuing description.